Although prolonged IL-12 treatment has been shown to increase the number of peripheral blood CD8' T cells expressing memory phenotypic markers, the antigenic specificity of these cells has not been examined. One of the objectives of this application is to perform serial ELISPOT and tetramer analyses to determine if systemic IL-12 treatment affects the number or phenotype of circulating CD8+ T cells capable of recognizing defined melanoma peptide antigens in vitro. We have shown that the induction of IFNgamma, a critical determinant of IL-12-induced tumor regression, progressively declines with successive IL-12 injections and the extent of this down modulation correlates negatively with clinical outcome. Although this apparent desensitization to IL-12 is most likely an innate physiologic mechanism designed to restrict the duration and severity of TH 1 immune responses, it may also limit the therapeutic utility of IL-12 as an antitumor agent. The elucidation of the molecular mechanism underlying the failure to sustain IL-12-induced IFNgamma production is therefore a priority and one of the objectives of this application. Preliminary data suggest that this failure cannot be attributed to a disappearance of cells expressing IL-12 receptors or a global loss of STATs in PBL. The studies proposed in this application will evaluate events downstream of receptor engagement such as the ability of IL-12 and other cytokines to induce STAT phosphorylation in vitro in PBL obtained at various time points throughout the course of IL-12 treatment. The studies will also assess the effects of IL- 12 treatment on the expression of inhibitors of cytokine signaling (e.g. the Suppressor of Cytokine Signaling or SOCS family members) and on the regulable members of Janus Kinase (JAK) family. Finally, since the limited production of IFNgamma by TH2 and virus-infected T cells has been shown to be secondary to hypermethylation of the IFNgamma promoter, we propose to evaluate the state of methylation of this promoter in PBL obtained serially throughout the course of IL-12 treatment. Collectively, these studies may provide insights into the regulation of IL-12 responses and offer clues as to how the antitumor activity of this important cytokine might be enhanced.